Micro Sampling and Testing

United States Department of Agriculture
Food Safety and Inspection Service
Understanding Microbiological
Sampling and Testing
FSIS 2014 EIAO Education Program
Philip Bronstein
Office of Public Health Science
Science Staff
Microbiological Issues Team

Todays Presentation
FSIS and industry testing activities
Sampling methods and design
Testing methods
Fitness for purpose
Validation
Issues specific to pathogen testing
Quantitative testing
Laboratory accreditation and communications
2

FSIS and Industry

Testing Programs

Microbiological Testing by FSIS

Laboratories
3 Field Service Labs administer regulatory testing programs
Routine monitoring, follow-up and
baseline study programs:
- Athens, Georgia
Executive Associate
EFSL-routine/other testing
LQAD-quality assurance
FERN- biosecurity
- St. Louis, Missouri
MWFSL-routine testing
- Alameda, California
WFSL-routine testing
4

FSIS Sampling Programs

Sample location
#
Collected
Domestic Federal
78,671
In commerce
Import
856
3,209
Fiscal year 2010 data (Oct 2009-Sept 2010), accessed 2/2/11

FSIS Sampling Program Objectives

Assess effectiveness of industry process controls
Provide critical feedback to industry
Monitor compliance with performance standards, zero
tolerance policies
Allow FSIS to monitor industry-wide trends
Serve as a strong incentive to reduce the occurrence of
pathogens in products
Capture pathogen serotype as well as PFGE patterns for
evaluation in PulseNet
6

FSIS Sampling Programs

Sampling plans measure compliance with performance
standards:
Salmonella and Campylobacter j/c/l verification programs
(400 mL rinsate, 2 x 50 sq cm sponge per pathogen)
Zero tolerance policies for food pathogens
E. coli O157:H7 (325 grams raw product),
Lm (25 grams RTE product, presence in food contact
surface swab, 25 grams pasteurized egg product)
Salmonella (325 grams RTE product or 100 grams
pasteurized egg product)
New methods: non-O157 Shiga toxin producing E. coli
7

Microbiological Testing by
FSIS-Regulated Establishments (Industry)
Fulfill regulatory requirement (9 CFR 310.25, 381.94,
430.4, 590.580)
Support decisions made in hazard analysis (9 CFR
417.2 (a))
On-going verification of HACCP plan (9 CFR 417.4
(a)(2))
Evaluate effectiveness of sanitary SOPs (9 CFR 416.14)
Fulfill purchase agreements
Respond to process deviations

Establishment Responsibilities For

Laboratory Testing
The establishment is ultimately responsible for the
testing they request from private laboratories
Has the establishment properly conveyed testing needs?
e.g., test portion equivalent to FSIS as opposed to the
default 25-g in protocols.
Is the laboratory aware of FSIS expectations?
Directives, Notices and guidance (some are pending)
Establishment should document detailed methodology
and validation information for FSIS review.
9

FSIS Verification of
Establishment Sampling and Testing
Programs
Effectiveness verified by FSIS
Performed by EIAOs during FSA
Establishment provides documentation
Technical and policy support provided through
AskFSIS
Establishment, not lab, is responsible for implementing
effective program
10

FSIS Verification of
Establishment Sampling and Testing
Programs
Focus of FSIS evaluation
Is the method fit for the intended purpose?
Does the method support the hazard analysis
decisions?
Is the method comparable to the appropriate FSIS
method (or is there justification for an alternative)?
Is a comparable or appropriate test portion used?
Is the method validated and used under validated
conditions?
Does the laboratory assure the quality of the results?
11

Assessing Sampling Plans
12

Why are pathogens hard to detect?
They are typically not evenly distributed

They are often injured when found in the product
They are able to cause disease at low levels
Detection may be inhibited by material in the
food product
13

Sampling Methods and Tools

Destructive sampling (e.g., RTE, ground
products, egg products)
Non-Destructive sampling
Pro when destructive sampling not an option
Examples:
Chicken carcass rinsate, carcass sponge samples
Food contact surface/Environmental sponge
sample
14

Sampling
All sampling plans have significant limitations
Therefore, we evaluate relative rigor of the program
Best sampling plans provide the opportunity but no
guarantee of detection
i.e., scattered contamination is difficult to detect
Frequent sampling and sampling multiple sites/time
points provides a better opportunity for detection
Examples:
Multiple samples per day vs. once per month
n60 per lot vs. one grab sample per lot
Does the type of sampling meet the intended need?
Destructive vs. non-destructive sampling
15

Sampling Plans
Statistical sampling plans assume
Uniform manufacturing conditions
Equal probability of contamination throughout the lot
(homogeneous distribution)
Independent, random sampling (equal probability of
sampling throughout the lot)
16

E. coli O157:H7 Contamination in a n60 Sampled Lot

(illustration)
1
10
Hot Spot
Sporadic/Background
17

E. coli O157:H7 Contamination in Ground Beef

(illustration)
100
<5
<5
40
30
<10
% contaminated samples
Combo bins
40% of product contaminated

by hour 3 of production
slug
0
1
Time of production, hrs
18

What is n60?
n60 = number of samples (n) = 60
Multiple representative samples provides best option
for detecting scattered contamination
Provides 95% confidence that no more than 5% of
food pieces the size of each n in the entire lot are
contaminated
Keys to success
Must ensure that sampling is as representative as
possible across the lot
Large composite n60 samples typical need a larger
test portion
19

Common Sampling Problems

Small sample or sampling method may not be ideal for
detection
Examples: small swab device, small carcass or
environmental area sampled
Sanitizer or excessive intervention might interfere with
the test
Insufficient drip time prior to carcass rinse procedure
Temperature abuse for the sample prior to testing
Holding under refrigeration for long periods allows
competing bacteria to grow
Freezing can kill some pathogens (e.g.,
Campylobacter)
20

Assessing Testing Methods
21

Key Players For Ensuring

Robust Testing Methods
The establishment that needs the testing
The laboratory they hire
The manufacturer of the screening test
they use
The organization validating the screening
test
22

Steps in Detection Methods
Sample collection
Sample preparation
Enrichment for the pathogen
Screening of the Pathogen
Confirmation of the Pathogen
23

Considerations for Testing Methods

Is the method fit for the intended purpose
of the analysis?
Has the method been optimized and
experimentally validated for sensitive
detection of pathogens?
Is the laboratory complying to the
validated method protocol?
24

Assessing Fitness For Purpose

Is the test portion appropriate to meet the
need?
Is the method enrichment-based with the
intent to detect the lowest possible
numbers of stressed pathogen cells?
Are confirmation procedures appropriate
for determining true negative samples?
25

The Test Portion

Laboratory sample preparation => test portion
a.k.a., analytical unit or analytical portion
Definition- the part of the sample that is actually
tested by the laboratory.
The test portion determines the theoretical (i.e.,

best possible) sensitivity of the test
i.e., 1 cell/test portion
25-gram- detecting 0.04 cells/gram is possible
325-gram- detecting 0.003 cells/gram is possible
26

Enrichment
Test portion is incubated 8-48 hours in a culture broth
Why?
Contamination levels are too low for detection
without enrichment
Must grow to high levels so very small volumes
have enough for later detection steps.
Different pathogens require a different broths
One vs two-stage enrichment
resuscitation vs selective growth
27

Considerations for proper enrichment

Resuscitation (lag phase) can require 2-3 hours before
log-phase growth begins
Some samples support slower growth
Has enrichment broth been tempered to warm
temperature prior to incubation?
Particularly critical for large test portions or shorter
incubation periods
28

Pathogen Growth During Enrichment

Log pathogen level
(e.g., cfu, MPN/gram)
10
logarithmic
lag
stationary
death
immunoassay
Possible
Loss of
Sensitivity
Prior to
confirmatory
retesting
PCR
4
0
2
24
Incubation time, hrs
29

Enrichment Period
Different screening tests require different levels of
enriched pathogen.
Shorter incubation periods (<15 hours) may warrant
additional scrutiny of laboratory compliance to the
validated protocol
Has enrichment/screening combination been validated
for a larger test portion?
Particular concern for large test portions incubated for
shorter periods
e.g., 375-gram test portion incubated for 8 hours
Proposed incubations < 8 hours may warrant OPHS
review
30

Role of Enrichment
31

Validation of Methods
32

Value of Validation
Determines performance characteristics of the method in
comparison to a gold standard method (i.e., usually FSIS
or FDA method)
Independent evaluation provides credibility
Rigor varies (multilab vs. single lab, # tests, etc)
Still must consider fitness for purpose and how the
method is applied
e.g., some AOAC-validated methods are not
consistent with FSIS goals or Compliance Guidelines
33

Method Validation
Recognized independent method validation
organizations:
Government: FSIS (MLG) and FDA (BAM)
AOAC International (U.S.A.)
AOAC Official Method (OM) validations
AOAC-RI Performance Tested Method
validations
AFNOR (France)- e.g., bioMerieux-Vitek tests
Others (ISO, MicroVal, NordVal, etc.)
However, past validations conducted by these
organizations may not be relevant to larger test portions
or other testing scenarios
34

Process for Validating

Qualitative Pathogen Methods
Series of laboratory experiments using
inoculated samples under controlled conditions.
Inoculate portions with pathogen strain at very
low level where only 20-80% of samples are
positive (i.e., fractional recovery)
Statistically compare percent of positive samples
in alternative method to reference method (FSIS
MLG)
35

Considerations
for Validation Data
Was method compared to an appropriate
reference method (e.g., FSIS MLG)?
If not performed by AOAC, AFNOR, etc.,
is supplemental validation data available?
May require additional scrutiny
36

Testing Method Specifications

Sensitivity: probability that truly positive samples are detected
as positive by analytical test
100 false negative rate
Specificity: probability that truly negative samples detected as
negative by analytical test
100 false positive rate
Level of detection (LOD): lowest level of contamination
reliably detected by analytical test
LOD expressed as ratio of organisms to quantity tested
material (e.g., CFU per gram, MPN per mL, CFU per
square-ft) but definitions vary (e.g., LOD50, POD)
37

Factors Impacting Detection

and Method Specifications
Detection as measured by sensitivity, false
negative rate and LOD can vary based on:
Specific strains of pathogen
Intrinsic factors for the sample matrix
Levels of competing bacteria
Fat, salt, pH and additives
Experimental design for the validation study
(e.g., cell stress, etc.)
38

Confirmatory Testing
Non-culture confirmation (e.g.,PCR)
Culture confirmation (e.g., FSIS confirmation)
Plating the enrichment on selective and differential
agar media
Immunomagnetic separation (IMS) necessary prior
to plating for E. coli O157:H7
Suspect colonies = presumptive positive
Purification and confirmatory identification tests
including:
Biochemical (e.g., identifies E. coli)
Serological (e.g., identifies O157 and H7)
Genetic (e.g., identifies stx = Shiga toxin genes) 39

Concerns for Confirmation

Do not re-sample the lot or sample
reserve!
Non-culture confirmation
Same considerations as the screening test
Used under validated conditions
Transport and storage of enrichment
Culture confirmation- carefully assess!

40

Complying To The
Validated Protocol
AOAC/AFNOR citations often do not
match the protocol in use.
Modifications are common, and some
contribute to greater potential for false
negative result.
Compare the lab procedure to the
validated protocol.
41

Methods Not Validated By

Recognized Organizations
Supplemental or extension validations
E. coli O157:H7 testing for 325-375g test
portions.
Modifications required for AOAC validated
procedures based on 25g
Instructions may not be clear for the lab
Non-O157 STEC No Objection Letter process
42

STEC Testing
Includes:
E. coli O157:H7
Six Non-O157 Shigatoxigenic E. coli
43

E. coli O157:H7 Analysis (MLG Chap. 5A)

Day 1
Sample Prep and Primary Enrichment

42C1 for 15-22 hours
Day 2
Perform PCR
All samples that do not test
PCR negative are carried
forward for further analysis
Day 2 cont.
Enrichment
confirm (-),
potential (+)
Screening
Immunomagnetic Bead Capture

& Rainbow Agar Plating
Day 3
O157 Latex Agglutination &

Sheep Blood Agar Plating
Day 4
Examine for Control

Bioluminescence & H7
Agglutination
Day 4 cont.
ELISA Shiga Toxin Assay
Day 4 cont.
Biochemical Identification
presumptive (+)
Confirmation
confirm (- ,+)
44

Non-O157 STEC Program

STEC = Shigatoxigenic E. coli
Six Non-O157 serotypes are targeted (O26,
0111, O103, O45, O121, O145)
Serotype strain must have stx (Shiga toxin)
and eae (intimin) genes
Methods are not well established and continue
to emerge and evolve (e.g., AOAC/AFNOR
validations beginning to emerge)
Temporary program for No Objection
45

Larger E. coli O157:H7 and NonO157 STEC Test Portions

Larger test portions (325-375 grams) are most important
for n60 and other composite samples containing many
samples
Less important for single grab samples of ground beef
final product testing when:
Trim and components have already been tested using
robust sampling and 325-375-gram test portions
multiple samples are collected throughout the
production day
Methods must be adapted, optimized and validated for
46
effective use with 325-375 gram test portions

E. coli O157:H7 and Non-O157 STEC

Testing Concerns
Supplemental validation and special instructions
for testing larger test portions.
For enrichment periods <15 hours, 325-375g
test portions typically often require longer
minimum enrichment period than 25g.
Culture-based detection and confirmation
requires immunomagnetic separation (IMS).
47

Listeria Testing
Includes:
L. monocytogenes testing (FSIS)
Listeria-like or Listeria spp. testing (industry)
48

Listeria monocytogenes (MLG Chap. 8)

Day 1
Day 2
PCR Method
Enrichment
Stomach 25g sample + 225 ml LEB

Incubate 20-24 hrs @ 30C
Secondary Enrichment
Inoculate 0.1ml to MOPS-BLEB
Incubate 18-24hrs at 35C
Day 3
Perform BAX PCR

Streak all PCR positive samples to MOX
Day 4
Pick Typical Colonies

Pick 20 colonies and collectively streak
for isolation on HBO
Day 5
Streak isolated colony to HBO
Day 6
Perform biochemical testing and

Inoculate CAMP test
Streak HBO plate
Day 7
Perform Ribosomal RNA based testing
Screening
confirm (-)
Confirmation
presumptive (+)
confirm (-/+)
49

Expectations For Listeria

Environmental Testing Equivalence
Compliance Guidelines, May 2006, pp. 42-44
For optimal sensitivity of detection, method for food
contact surface testing must:
validated by a recognized body (e.g., AOAC, AFNOR)
be enrichment-based
enrich the entire sponge/swab sample
i.e., aliquot from sponge/swab does not provide
opportunity to detect bacteria trapped in the
sponge.
50

Analytes for Industry Food Contact

or Environmental Surface Testing
Production establishment laboratories test for one of the
following:
Listeria monocytogenes: Use internationally recognized
enrichment-based method that biochemically confirms
culture as L. monocytogenes
Listeria spp.: Use internationally recognized enrichmentbased method that uses ELISA, PCR or other screening
technology to provide more rapid but less specific Listeria
spp. result
Listeria-like indicator bacteria: Use the first part of an
internationally recognized enrichment-based method to
find suspect Listeria colonies (e.g., darkened colonies on
MOX using the FSIS method)
51

Salmonella Testing
Raw products
Meat and turkey carcass sponge samples
Chicken carcass rinsates
Raw ground meat and poultry
Processed products
RTE (325g portion)
Pasteurized egg
52

Salmonella (MLG Chap. 4)

Day 1
Enrichment
Stomach sample + BPW

Incubate 20-24 hrs at 35C
Perform PCR
Day 2
All samples that do not test PCR

negative are carried forward to RV and
TT broth
Day 3
Streak RV and TT on BGS and

DMLIA plates
Day 4
Pick suspect colony from Plate

medium to TSI and LIA slants.
Incubate slants with loosened caps
for 22-24 hrs at 35C
Day 5
Perform O and H serology on

slants. Streak on SBA for
biochemical testing
Day 6
Perform biochemical testing using

colony from SBA plate.
Screening
confirm (-)
Confirmation
presumptive (+)
confirm (-/+)
53

Campylobacter Testing
Qualitative or quantiative
Semi-quantitative for regulatory application
Target = C. jejuni, C. lari or C. coli
54

Campylobacter (MLG Chap. 41) - Quantitative

Day 1-2
Day 3
Direct Plating
(no enrichment)
Direct plating onto Campy-Cefex

Incubate 48 hrs at 42C
Count colonies
Pick 5 typical colonies
confirm (-)
Confirmation
Microscope examination for

morphology/motility
Latex agglutination
confirm (-/+)
RESULTS ARE REPORTED AS POSITIVE/NEGATIVE

1 CFU = POSITIVE
55

Campylobacter (MLG Chap. 41) - Qualitative

Day 1-2
Enrichment
Stomach sample + BF-BEB

Day 3
Direct plating onto Campy-Cefex

Day 5
Count colonies
Pick 5 typical colonies
Direct Plating
(no enrichment)
confirm (-)
Confirmation
Microscope examination for

morphology/motility
Latex agglutination
confirm (-/+)
RESULTS ARE NOT USED FOR REGULATORY PURPOSES

56

Issues for
Campylobacter Testing
Campylobacter is highly vulnerable to
freezing
Do not freeze samples
Can be a challenging test (inconsistent

results across labs)
57

Quantitative Testing
NOTE- Quantitative testing typically cannot
accommodate larger test portions and provide
the opportunity for detection that a qualitative
test can provide.
Two options:
MPN
Direct plating
58

Most Probable Number (MPN)

Enumeration Analysis
Traditional enrichment-based analyses are performed on
three or more dilutions, each typically in triplicate, from a
single sample homogenate (i.e., MPN = method format,
not a specific method per se)
Advantages:
Better sensitivity (lower LOD) than direct plating
Disadvantages:
Very resource intensive/expensive
Test portion 3.3 grams (FSIS method = < 33 grams)
Application:
For quantifying low levels of pathogens (e.g.,
Salmonella, E. coli O157:H7, L. monocytogenes)
59

Quantitative Testing
MPN (most probable number)
325 grams
+ 10 fold buffer
= 0.1 grams/mL
10 mL
(1 gram x 3)
10 mL 1:10
(0.1 gram x 3)
10 mL 1:100
(0.01 gram x 3)
Dilute 1:10, 1:100

enrich
+++
enrich
enrich
-++
--+
60

Example:
3-2-1 = Y MPN/g
(use MPN table to determine Y)
Total tested:
3.33 grams
(33 grams FSIS method)
Level of Detection =
< 0.3 MPN/gram (0-0-0)
<0.03 MPN/gram (FSIS method)
61

Direct Plating Enumeration Methods

Product is homogenized in diluent and small volume is
directly dispensed onto agar media (i.e., sometimes
there is a 1-2 h resuscitation step, but enrichment is
never used prior to plating)
Advantages:
Allows easy inexpensive quantitative analysis
Disadvantages:
Accommodates only a very small test portion
Higher limit of detection (i.e., often 100 CFU/g) not
suitable for detecting low levels of pathogens
Application:
Expedient for higher level analytes (e.g., indicators,
Campylobacter, S. aureus, C. perfringens, B. cereus)
62

Quantitative Testing: Direct Plating

CFU (colony forming unit)
325 grams
+ 10-fold buffer
= 0.1 grams/mL
1 mL
(0.1 gram)
Dilute 1:10, 1:100
No
enrichment
6 cfu/1 mL/0.1 g
= 60 cfu/g
1 mL 1:10
(0.01 gram)
1 mL 1:100
(0.001 gram)
Total tested 0.11 grams
Level of Detection = <10 cfu/gram (0 cfu from homogenate)
63

The Establishment
And The Laboratory
64

Establishment and Laboratory

Communication is Vital
The communication challenge
The establishment may not understand the
testing conducted on their behalf.
The laboratory does not necessarily know
what the establishment needs.
The laboratory may not be aware of special
validated procedures for larger test portions.
The establishment is ultimately responsible
65

Does the establishment have the

necessary documentation?
Can the establishment provide the method used for
microbial detection?
Can the establishment provide evidence that the method
used was properly validated by an independent body?
Can the establishment explain why the method fits the
need?
66

Issues for Industry Labs

On-site vs. off-site labs
Shipment of samples/handling during shipment
Overarching concerns for on-site labs
Is testing effective?
Is testing safe in that facility?
Enrichment of pathogens in an establishments
Evaluate the following:
Are personnel qualified?
Does the lab have proper equipment and materials for
testing and disposal of contaminated media?
Do they follow the validated testing protocol?
67

ISO 17025 Laboratory Accreditation

ISO 17025 = protocol for establishing and documenting
a microbiology laboratory quality program (i.e., HACCP
for labs)
Accrediting bodies = A2LA and others
Accreditation implies robust quality program but does not
necessarily indicate methods meet FSIS expectations.
68

Helpful Guidance
69

Existing Agency Guidance

Compliance Guides
Compliance Guidelines for the Interim Final Rule, "Control Of
Listeria monocytogenes In Ready-To-Eat Products (May
2006)
Compliance Guideline for Controlling Salmonella and
Campylobacter in Poultry (Third Edition, May 2010)
Draft Compliance Guideline for Sampling Beef Trimmings for
Escherichia coli O157:H7 (August 2008)
Establishment Guidance for the Selection of a Commercial or
Private Microbiological Testing Laboratory (March 2012)
FSIS Guidance for Evaluating Test Kit Performance (October
2010)
Evaluation of Microbiological Methods Used by
Establishments (FSIS Directive 5100.1 Rev. 3, Attachment 1)
70

Existing Agency Guidance - askFSIS

Method modifications (AskFSIS Q&A, 5/19/09)
Sensitivity and specificity (AskFSIS Q&A, 5/19/09)
Validation by independent organizations (AskFSIS Q&A,
5/19/09)
Confirming E. coli O157:H7 Screen Positive Test Results
(AskFSIS Q&A, 4/26/10)
E. coli O157:H7 Test Portions Smaller than 325 grams
(AskFSIS Q&A, 8/3/2010)
71

Future Guidance
Selecting a private laboratory
Laboratory best practices
List of validated methods
NOTE- Not necessarily fit for purpose
Future askFSIS Q&As
72

Questions?
Ask me now
or for future questions:
Enter question into askFSIS
Provide documentation for review
Request Sampling Queue
73

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United States Department of Agriculture

Food Safety and Inspection Service

United States Department of Agriculture

United States Department of Agriculture

FSIS and Industry

United States Department of Agriculture

Microbiological Testing by FSIS

United States Department of Agriculture

FSIS Sampling Programs

Fiscal year 2010 data (Oct 2009-Sept 2010), accessed 2/2/11

United States Department of Agriculture

FSIS Sampling Program Objectives

United States Department of Agriculture

FSIS Sampling Programs

United States Department of Agriculture

United States Department of Agriculture

Establishment Responsibilities For

United States Department of Agriculture

United States Department of Agriculture

United States Department of Agriculture

Assessing Sampling Plans

United States Department of Agriculture

Why are pathogens hard to detect?

They are typically not evenly distributed

United States Department of Agriculture

Sampling Methods and Tools

United States Department of Agriculture

United States Department of Agriculture

United States Department of Agriculture

E. coli O157:H7 Contamination in a n60 Sampled Lot

United States Department of Agriculture

E. coli O157:H7 Contamination in Ground Beef

40% of product contaminated

Time of production, hrs

United States Department of Agriculture

United States Department of Agriculture

Common Sampling Problems

United States Department of Agriculture

Assessing Testing Methods

United States Department of Agriculture

Key Players For Ensuring

United States Department of Agriculture

Steps in Detection Methods

United States Department of Agriculture

Considerations for Testing Methods

United States Department of Agriculture

Assessing Fitness For Purpose

United States Department of Agriculture

The Test Portion

The test portion determines the theoretical (i.e.,

United States Department of Agriculture

United States Department of Agriculture

Considerations for proper enrichment

United States Department of Agriculture

Pathogen Growth During Enrichment

Incubation time, hrs

United States Department of Agriculture

United States Department of Agriculture

United States Department of Agriculture

United States Department of Agriculture

United States Department of Agriculture

United States Department of Agriculture

Process for Validating

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